Mass spectrometry has developed into a useful tool to the biomedical community. Mass spectrometers used in biological applications primarily utilize either electrospray ionization (ESI), or matrix assisted laser desorption/ionization (MALDI), for transferring the molecules into a vacuum chamber. The mass analysis is typically accomplished using electrostatic or magnetic methods, or by time-of-flight (TOF) analysis.
Differences between the techniques arise from the methods used to generate the molecules in their ionic form. In ESI, the material is transferred to the high vacuum chamber while maintaining the ionic form that prevails in solution. In MALDI the laser desorption of the underlying matrix transfers molecules to the vacuum in either their neutral or ionic form. It has been estimated that only one out of 104 molecules enter the vacuum as ions. Nevertheless, because the ion detector is extremely sensitive, the small fraction of molecules that do enter the vacuum as ions are easily detected and measured.
It is known in the art that photoionization techniques exist wherein lasers are employed simply to ionize the materials. While those techniques may operate satisfactorily for some applications, and have been exploited for commercial instruments, they are infrequently used by the biological community because they lack a convincing inherent advantage. A need thus exists to provide significant new information that cannot be obtained by other ionization schemes.
A need also exists to provide researchers with an inexpensive and rapid indication of the three dimensional shape of molecules and clusters of molecules.
As an example, the analysis of proteins by two dimensional (2D) gel electrophoresis coupled to mass spectrometry is insensitive to equivalent modifications of a protein that can occur at several sites. For example, stathmin, a tubulin binding protein, has several sites of phosphorylation. While isoelectric focusing and mass spectrometry can separate the non-, mono-, di-, tri-, and tetra-phosphorylated forms, neither method can differentiate forms of the protein that are monophosphorylated, but at different sites.
Further by example, the formation of correct disulfides in proteins are often required for proper function and protease resistance. Proteins with several cysteines in the amino acid sequence can have multiple forms due to cross-linking of the disulfides. The formation of incorrect disulfides can result in changes in the tertiary structure with consequent loss of enzymatic activity, or can result in changes in protein-protein binding. Unless the formation of non-native disulfides causes gross structural changes these alternative forms are not readily separable. It would be useful, therefore, to have available a shape-sensitive mass spectrometry technique that could readily resolve and quantitate the different protein forms due to alternative disulfide formation, or modification at each of a number of different sites.
In addition to the large molecules such as proteins, molecular clusters are an important cornerstone of nanoscale science. This importance derives from both their intrinsic interest, as well as their role as precursors in the production of cluster-assembled materials. Examples of clusters include atomically pure clusters such as those of carbon, which have achieved significant attention due in large part to an important member of the group, buckminsterfullerene. In addition there are clusters composed of metal atoms, rare gas atoms and metal oxides. In addition, there are composite clusters consisting of molecules with other molecules, and many combinations of rare gas or metal atoms are known.
Of particular interest to this invention are metallic clusters, composed of up to 40 atoms of transition metals such as nickel, palladium, or platinum. Such metallic clusters exhibit interesting structural and photophysical properties and, in addition, are important catalysts in many chemical reactions.
It is important to note that even relatively simple clusters exhibit a large number of stable isomers. Model studies suggest, for example, that the number of stable isomers grows exponentially with cluster size, reaching on the order of 1021 stable isomers for 55 atom Lennard-Jones systems. While not all of the theoretically calculated cluster isomers are necessarily present in a typical cluster source, studies have shown that even for small cluster numbers several isomeric forms have significant likelihood of being present.
Prior to this invention, however, there has not existed suitable instrumentation for identifying and segregating the different isomeric cluster forms. While mass selection of clusters is routine, the separation of isomers having the same or approximately the same mass, and the further identification and separation of clusters exhibiting isomer-specific properties, has not been accomplished in a satisfactory manner.
The importance of this need is significant. For example, both theoretical investigations and experiments have shown the catalytic function of a cluster to depend strongly on the number of atoms in the cluster. For example, in one study of the catalytic effectiveness of palladium clusters in the polymerization of acetylene, it was found that Pd6 clusters produce the highest selectivity for generating butadiene, whereas butene is formed with the highest selectivity by the Pd20 clusters. It is thus apparent that the structure of the cluster is closely correlated to its catalytic activity: certain cluster sizes have specific structures with certain exposed faces and steps. Given the large number of isomeric cluster forms that are possible, even for modest atom numbers, it is clear that the measured selectivities are averages over all cluster shapes in the sample. This suggests that the selectivity of the catalytic processes could be greatly enhanced if, in addition to the cluster mass, one were able to isolate specific cluster forms. Moreover, the possibility of assembling metastable species to form extended systems with novel physical or chemical properties makes the study of higher-energy cluster conformers of significance to the broader nanoscience community.
A typical cluster source generates a wide distribution of cluster masses and cluster structures. While it is straightforward to use mass spectrometric techniques to separate cluster masses, no technique existed, prior to this invention, to select specific cluster structures. In order to separate specific cluster shapes out of a mixture containing a large number of sizes and isomers, what is needed is an ability to exploit a property that allows the identification of one isomer vis-à-vis all the other isomers.